The present invention concerns amino acid derivatives possessing aspartyl protease inhibitory properties, in particular Nxcex5-amino acid substituted L-lysine derivatives (and analogs) possessing aspartyl protease inhibitory properties. It describes also the synthetic methodology used to make these derivatives and their biological applications. In addition, this invention relates to different pharmaceutical compositions comprising these compounds. The compounds and the pharmaceutical compositions of this invention have been shown to inhibit the activity of HIV aspartyl protease, an enzyme essential for virus maturation. The inhibitory property may be advantageously used to provide compounds with antiviral properties against HIV viruses, including the HIV-1 and HIV-2 viruses.
The HIV (human immunodeficiency virus) retrovirus is responsible for causing the disease known as AIDS (acquired immunodeficiency syndrome). HIV infection is characterized by a period immediately following infection, called asymptomatic, which is devoid of clinical manifestations in the patient. Progressive HIV-induced destruction of the immune system then leads to increased susceptibility to opportunistic infections, which eventually produces a syndrome called AIDS-related complex (ARC) characterized by symptoms such as persistent generalized lymphadenopathy, fever, weight loss, followed itself by full blown AIDS.
As the first step of its replication cycle, the HIV-1 retrovirus attaches primarily to the CD4 receptor (a 58 kDa transmembrane protein) to gain entry into susceptible cells, through high-affinity interactions between the viral envelope glycoprotein (gp 120) and a specific region of the CD4 molecule found in CD4 (+) T-helper lymphocytes and other cells carrying the receptor (Lasky L. A. et al., Cell vol. 50, p. 975-985 (1987)). The HIV genetic material, in the form of RNA, is then transcribed into DNA by a viral enzyme carried by the virus called reverse transcriptase. The viral DNA now called provirus is then transported into the cell nucleus in the form of a preintegration complex and attached to the cell DNA by another viral enzyme called integrase. Following integration, the viral DNA then serves as a template for viral gene expression by the host transcription system. The primary RNA transcripts made from the provirus are synthesized by the host cell RNA polymerase II whose activity is modulated by two virus-encoded proteins, Tat and Rev. The viral proteins are expressed mainly in the form of polyproteins. After the infected cell has produced all the different HIV polyproteins and genetic material, they assemble at the cell membrane and are released from the cell in the form of an immature viral particle. A third viral enzyme known as protease then cleaves the polyproteins to give the mature, infectious viral particle. The polyproteins that are cleaved by the HIV protease are the Gag and Gag-Pol precursors, whose cleavage gives rise to structural proteins and viral enzymes.
A number of synthetic antiviral agents have been designed to block various stages in the replication cycle of HIV, although only those developed against the viral enzymes have reached the market so far. The latter include compounds which block viral reverse transcriptase (for example, didanosine and zidovudine (AZT)), or the viral protease (for example, ritonavir and indinavir). Although these drugs have improved significantly the survival time and quality of life of AIDS patients, the administration of most of these agents leads to unwanted side effects, such as anemia, neurotoxicity and bone marrow suppression.
Anti-protease compounds represent the most recent drugs developed to block HIV replication. These compounds inhibit the formation of infectious virions by interfering with the processing of viral polyprotein precursors by the viral protease. The antiviral potential of HIV protease inhibitors was first demonstrated using peptidic inhibitors. Such peptidic compounds, however, are typically large and complex molecules that tend to exhibit poor bioavailability and limited stability in the body. New compounds devoid of these drawbacks are urgently needed to treat HIV infections. In addition, mutations arising during HIV replication lead to resistance to the currently available protease inhibitors, so new compounds with original structure are desirable to fight these resistant viral strains.
The present invention provides a novel class of compounds, including their pharmaceutically acceptable derivatives. These compounds have an affinity for aspartyl proteases, in particular, HIV aspartyl protease. Therefore, these compounds are useful as inhibitors of such proteases. These compounds can be used alone or in combination with other therapeutic or prophylactic agents for the treatment or prophylaxis of viral infection.
According to a preferred embodiment, the compounds of this invention are capable of inhibiting HIV viral replication in human CD4+ T-cells, by inhibiting the ability of HIV aspartyl protease to catalyse the hydrolysis of peptide bonds present in viral Gag and Gag-Pol polyproteins. These novel compounds can thus serve to reduce the production of infectious virions from acutely and chronically infected cells, and can inhibit the initial or further infection of host cells. Accordingly, these compounds are useful as therapeutic and prophylactic agents to treat or prevent infection by HIV-1 and HIV-2, which may result in asymptomatic infection, AIDS-related complex (ARC), acquired immunodeficiency syndrome (AIDS), AIDS-related dementia, or similar diseases of the immune system, and related viruses such as HTLV-I and HTLV-II, and simian immunodeficiency virus.
It is the main objective of this invention to provide a novel class of molecules that are aspartyl protease inhibitors, and particularly, HIV aspartyl protease inhibitors.
The present invention relates to a class of Nxcex5-amino acid substituted L-lysine derivatives (including its lower and higher homologues and analogs) as well as their pharmaceutically acceptable derivatives (e.g. salts).
Accordingly, the present invention in accordance with one aspect thereof provides a compound of formula I 
a compound of formula II 
and when the compound of formula I and II comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein n is 3 or 4
wherein Y is, O, S or Nxe2x80x94CN
wherein Cx is selected from the group consisting of xe2x80x94COOM, COOR6, xe2x80x94CHO, xe2x80x94CH2OR7, xe2x80x94CH2OCOR8, xe2x80x94CONHR9 and xe2x80x94CONR10,R11, wherein M is an alkali metal (e.g. Na, K, Cs, etc) or alkaline earth metal (Ca, Mg, etc.),
wherein R1 is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R2 is selected from the group consisting of a benzenesulfonyl group of formula III, 
xe2x80x83and a thiophenesulfonyl group of formula IV, 
wherein R3 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, a phenyl or a benzyl group
wherein R4 is selected from the group consisting of H, a group of formula IIIa 
xe2x80x83a group of formula IVa 
xe2x80x83C6H11xe2x80x94, C5H10Nxe2x80x94CH2CH2xe2x80x94, OC4H8Nxe2x80x94CH2CH2xe2x80x94 (i.e. morpholine-4-CH2CH2xe2x80x94), C6H5CH2CH2xe2x80x94, 2,3-(CH3O)2C6H3CH2xe2x80x94, C6H5xe2x80x94, 2-C5H4N (i.e. 2-pyridinyl), 3-C5H4N (i.e. 3-pyridinyl), 4-C5H4N (i.e. 4-pyridinyl), 3-quinolyl, C6H5CSxe2x80x94, 2-naphthyl-SO2xe2x80x94 and a group of formula R4Cxe2x80x94COxe2x80x94, R4C being selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, (e.g. CH3xe2x80x94, iso-butyl, iso-propyl, tert-butyl, tert-butyl-CH2xe2x80x94), CF3, 1-pyrrolidinyl, 4-morpholinyl, tetrahydro-3-furanyloxy, 4-CH3OC6H4NHxe2x80x94, CH3NHxe2x80x94, HOCH2CH2NHxe2x80x94, 9-fluorenyl-CH2Oxe2x80x94, tert-butylO-, iso-butylO-, C6H5CH2Oxe2x80x94, CH3Oxe2x80x94, unsubstituted C6H5xe2x80x94, C6H5xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH, unsubstituted C6H5CH2xe2x80x94, C6H5CH2xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH, unsubstituted C6H5CH2CH2xe2x80x94, and C6H5CH2CH2xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R5 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 8 carbon atoms, (e.g. CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94), HOCH2xe2x80x94, C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6H5CH(OH)xe2x80x94, C6H5CH(CN)xe2x80x94, C6F5CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)-NHCH2xe2x80x94C6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94 (i.e. pyridine-2-CH2xe2x80x94), C5H4N-3-CH2xe2x80x94 (i.e. pyridine-3-CH2xe2x80x94), C5H4N-4-CH2xe2x80x94 (i.e. pyridine-4-CH2xe2x80x94), 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94, imidazole-4-CH2xe2x80x94, thiazole-4-CH2xe2x80x94 and substituted benzyl (e.g. benzyl substituted by a group being as defined for R12 below, e.g. 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-Oxe2x80x94C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94),
wherein Ra represents a member selected from the group consisting of 
xe2x80x83wherein Met is a methylene linked to the xcex1xe2x80x2 nitrogen
wherein R6 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms and glycyl
wherein R7 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms
wherein R8 is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R9 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, xe2x80x94OH, xe2x80x94NH2 and xe2x80x94CH2CH2OH
wherein R10 and R11 are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms
wherein m is 0 or 1
wherein o is 0 or 1
wherein R12 and R13 are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10 R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10and xe2x80x94CH2OH, R10 and R11 being as defined herein.
More particularly, this invention provides a compound of formula IA 
and when the compound of formula IA comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein n is 3 or 4
wherein Y is O, S or Nxe2x80x94CN
wherein Cx is selected from the group consisting of xe2x80x94COOM, COOR6, xe2x80x94CHO, xe2x80x94CH2OR7, xe2x80x94CH2OCOR8, xe2x80x94CONHR9 and xe2x80x94CONR10R11, wherein M is an alkali metal (e.g. Na, K, Cs, etc) or alkaline earth metal,
wherein R1 is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R2 is selected from the group consisting of a benzenesulfonyl group of formula III, 
xe2x80x83and a thiophenesulfonyl group of formula IV, 
wherein R3 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, a phenyl or a benzyl group
wherein R4 is selected from the group consisting of H, C6H11xe2x80x94, C5H10Nxe2x80x94CH2CH2xe2x80x94, OC4H8Nxe2x80x94CH2CH2xe2x80x94 (i.e. morpholine-4-CH2CH2xe2x80x94), C6H5CH2CH2xe2x80x94, 2,3-(CH3O)2C6H3CH2xe2x80x94, C6H5xe2x80x94, 2-C5H4N, 3-C5H4N, 4-C5H4N, 3-quinolyl, CH3COxe2x80x94, CF3CO, C6H5COxe2x80x94, C6H5CSxe2x80x94, 4CH3OC6H4CH2COxe2x80x94, C6H5CH2CH2COxe2x80x94, iso-butyl-COxe2x80x94, iso-propyl-COxe2x80x94, tert-butyl-COxe2x80x94, tert-butyl-CH2COxe2x80x94, 1-pyrrolidine-COxe2x80x94, 4-morpholine-COxe2x80x94, carbotetrahydro-3-furanyloxy, 4-CH3OC6H4NHCOxe2x80x94, CH3NHCOxe2x80x94, HOCH2CH2NHCOxe2x80x94, 9-fluorenylmethoxycarbonyl, tert-butylO-COxe2x80x94, iso-butylO-COxe2x80x94, C6H5CH2Oxe2x80x94COxe2x80x94, CH3Oxe2x80x94COxe2x80x94, C6H5SO2xe2x80x94, 4-CH3C6H4SO2xe2x80x94, 4-CF3C6H4SO2xe2x80x94, 4-NO2C6H4SO2xe2x80x94, 4-NH2C6H4SO2xe2x80x94, 4-AcNHC6H4SO2xe2x80x94, 4-FC6H4SO2xe2x80x94, 4-ClC6H4SO2xe2x80x94, 4-BrC6H4SO2xe2x80x94, 4-CH3OC6H4SO2xe2x80x94, 2-thiophene-SO2xe2x80x94 and 2-naphthyl-SO2xe2x80x94
wherein R5 is selected from the group consisting of H, CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94, HOCH2xe2x80x94, C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6F5CH2xe2x80x94, 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-Oxe2x80x94C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)NHCH2xe2x80x94C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94, C5H4N-3-CH2xe2x80x94, C5H4N-4-CH2xe2x80x94, 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94 and thiazole-4-CH2xe2x80x94
wherein R6 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms and glycyl
wherein R7 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms
wherein R8 is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R9 is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, xe2x80x94OH, xe2x80x94NH2 and xe2x80x94CH2CH2OH
wherein R10 and R11 are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms
wherein m is 0 or 1
wherein o is 0 or 1
wherein R12 and R13 are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH, R10 and R11 being as defined herein.
This invention also provides a compound of formula Ia 
and when the compound of formula Ia comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein Cx is selected from the group consisting of xe2x80x94COOM and xe2x80x94CH2OH, M being an alkali metal or alkaline earth metal,
wherein R1A is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R2A and R4A are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R5A is selected from the group consisting of H, a straight or branched alkyl group of 1 to 8 carbon atoms, (e.g. CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94, HOCH2xe2x80x94), C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6H5CH(OH)xe2x80x94, C6H5CH(CN)xe2x80x94, C6F5CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)-NHCH2xe2x80x94C6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94 (i.e. pyridine-2-CH2xe2x80x94), C5H4N-3-CH2xe2x80x94 (i.e. pyridine-3-CH2xe2x80x94), C5H4N-4-CH2xe2x80x94 (i.e. pyridine-4-CH2xe2x80x94), 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94, imidazole-4-CH2xe2x80x94, thiazole-4-CH2xe2x80x94 and substituted benzyl (e.g. benzyl substituted by a group as defined for R2A, e.g. 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-O-C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94),
and wherein R10 and R11 are as defined herein.
This invention also provides a compound of formula Ib 
and when the compound of formula Ib comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein Cx is selected from the group consisting of xe2x80x94COOM, and xe2x80x94CH2OH, M being an alkali metal or alkaline earth metal,
wherein R1A is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R2A and R4B are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R5A is selected from the group consisting of H, a straight or branched alkyl group of 1 to 8 carbon atoms, (e.g. CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94), HOCH2xe2x80x94, C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6H5CH(OH)xe2x80x94, C6H5CH(CN)xe2x80x94, C6F5CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)-NHCH2xe2x80x94, C6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94 (i.e. pyridine-2-CH2xe2x80x94), C5H4N-3-CH2xe2x80x94 (i.e. pyridine-3-CH2xe2x80x94), C5H4N-4-CH2xe2x80x94 (i.e. pyridine-4-CH2xe2x80x94), 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94, imidazole-4-CH2xe2x80x94, thiazole-4-CH2xe2x80x94 and substituted benzyl (e.g. benzyl substituted by a group as defined for R2A, e.g. 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-Oxe2x80x94C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94),
and wherein R10 and R11 are as defined herein.
This invention also provides a compound of formula Ic 
and when the compound of formula Ic comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein Cx is selected from the group consisting of xe2x80x94COOM and xe2x80x94CH2OH, M being an alkali metal or alkaline earth metal,
wherein R1A is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R2B and R4B are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R5A is selected from the group consisting of H, a straight or branched alkyl group of 1 to 8 carbon atoms, (e.g. CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94), HOCH2xe2x80x94, C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6H5CH(OH)xe2x80x94, C6H5CH(CN)xe2x80x94, C6F5CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)-NHCH2xe2x80x94C6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94 (i.e. pyridine-2-CH2xe2x80x94), C5H4N-3-CH2xe2x80x94 (i.e. pyridine-3-CH2xe2x80x94), C5H4N-4-CH2xe2x80x94 (i.e. pyridine-4-CH2xe2x80x94), 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94, imidazole-4-CH2xe2x80x94, thiazole-4-CH2xe2x80x94 and substituted benzyl (e.g. benzyl substituted by a group as defined for R2A, e.g. 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-Oxe2x80x94C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94),
and wherein R10 and R11 are as defined herein.
This invention also provides a compound of formula Id 
and when the compound of formula Id comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein Cx is selected from the group consisting of xe2x80x94COOM and xe2x80x94CH2OH, M being an alkali metal or alkaline earth metal,
wherein R1A is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R4A is selected from the group consisting of H, a straight or branched alkyl group of 1 to 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94OCH2C6H5, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R4C is selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, (e.g. CH3xe2x80x94, iso-butyl, iso-propyl, tert-butyl, tert-butyl-CH2xe2x80x94), CF3, pyrrolidine, 4-morpholine, tetrahydro-3-furanyloxy, 4-CH3OC6H4NHxe2x80x94, CH3NHxe2x80x94, HOCH2CH2NHxe2x80x94, 9-fluorenyl-CH2Oxe2x80x94, tert-butylO-, iso-butylO-, C6H5CH2Oxe2x80x94, CH3Oxe2x80x94, unsubstituted C6H5xe2x80x94, C6H5xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH, unsubstituted C6H5CH2xe2x80x94, C6H5CH2xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH, unsubstituted C6H5CH2CH2xe2x80x94, and C6H5CH2CH2xe2x80x94 substituted by one or more members (e.g. one or two) selected from the group consisting of F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR10R11, xe2x80x94NHCOR10, xe2x80x94OR10, xe2x80x94SR10, xe2x80x94COOR10, xe2x80x94COR10 and xe2x80x94CH2OH,
wherein R5A is selected from the group consisting of H, a straight or branched alkyl group of 1 to 8 carbon atoms, (e.g. CH3xe2x80x94, CH3CH2CH2xe2x80x94, CH3CH2CH2CH2xe2x80x94, iso-C4H9xe2x80x94, C6H11CH2xe2x80x94), HOCH2xe2x80x94, C6H5CH2OCH2xe2x80x94, benzyl-OCH(CH3), HO2CCH2xe2x80x94, HO2CCH2CH2xe2x80x94, NCxe2x80x94CH2xe2x80x94, H2NC(O)CH2xe2x80x94, H2NC(O)CH2CH2xe2x80x94, 4-CH3C6H4CH2SCH2xe2x80x94, CH3SCH2CH2xe2x80x94, H2NCH2CH2CH2CH2xe2x80x94, C6H5xe2x80x94, C6H5CH2xe2x80x94, C6H5CH(OH)xe2x80x94, C6H5CH(CN)xe2x80x94, C6F5CH2xe2x80x94, 4-(9-fluorenylmethoxycarbonyl)-NHCH2xe2x80x94C6H4CH2xe2x80x94, C5H4N-2-CH2xe2x80x94 (i.e. pyridine-2-CH2xe2x80x94), C5H4N-3-CH2xe2x80x94 (i.e. pyridine-3-CH2xe2x80x94), C5H4N-4-CH2xe2x80x94 (i.e. pyridine-4-CH2xe2x80x94), 2-thiophene-CH2xe2x80x94, indole-3-CH2xe2x80x94, 2-benzothiophene-CH2xe2x80x94, Nxcfx84-benzyl-imidazole-4-CH2xe2x80x94, imidazole-4-CH2xe2x80x94, thiazole-4-CH2xe2x80x94 and substituted benzyl (e.g. benzyl substituted by a group as defined for R2A, e.g. 4-tert-butyl-C6H4CH2xe2x80x94, 4-HOC6H4CH2xe2x80x94, 4-benzyl-Oxe2x80x94C6H4CH2xe2x80x94, 4-NO2C6H4CH2xe2x80x94, 2-FC6H4CH2xe2x80x94, 3-FC6H4CH2xe2x80x94, 4-FC6H4CH2xe2x80x94),
and wherein R10 and R11 are as defined herein.
In addition, this invention provides pharmaceutical compositions in which these novel compounds of formula I or II, (as well as of formula IA, Ia, Ib, Ic and Id) derived from L-lysine or L-lysine derivatives (as well as lower and higher homologues) are used to inhibit aspartyl proteases, including HIV aspartyl protease, thus providing protection against HIV infection.
Thus the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of at least one compound of formula I, formula II (as well as of formulae IA, Ia, Ib, Ic, and Id) and as applicable pharmaceutically acceptable ammonium salts thereof.
The terms xe2x80x9cHIV proteasexe2x80x9d and xe2x80x9cHIV aspartyl proteasexe2x80x9d are used interchangeably and refer to the aspartyl protease encoded by the human immunodeficiency virus type 1 or 2. In a preferred embodiment of this invention, these terms refer to the human immunodeficiency virus type 1 aspartyl protease.
The term xe2x80x9cpharmaceutically effective amountxe2x80x9d refers to an amount effective in treating HIV infection in a patient.
The term xe2x80x9cprophylactically effective amountxe2x80x9d refers to an amount effective in preventing HIV infection in a patient. As used herein, the term xe2x80x9cpatientxe2x80x9d refers to a mammal, including a human.
The terms xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d, xe2x80x9cpharmaceutically acceptable adjuvantxe2x80x9d and xe2x80x9cphysiologically acceptable vehiclexe2x80x9d refer to a non-toxic carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
The compounds of this invention include pharmaceutically acceptable derivatives of the compounds of formula I, formula II (as well as of formulae IA, Ia, Ib, Ic, and Id) and as applicable pharmaceutically acceptable ammonium salts thereof. A xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an antivirally active metabolite or residue thereof.
Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and Nxe2x80x94(C1-4 alkyl)4+ salts.
The compounds of this invention contain one or more asymmetric carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomer, diastereomeric mixtures and individual diastereoisomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be of the R or S configuration.
Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term xe2x80x9cstablexe2x80x9d, as used herein, refers to compounds which possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art. Typically, such compounds are stable at a temperature of 40xc2x0 C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of such acid salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylhydrogensulfate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycollate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthylsulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, perchlorate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate.
This invention also envisions the quaternization of any basic nitrogen containing groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
The compounds of this invention are readily prepared using conventional techniques from commercially available and inexpensive starting materials. The relative ease of synthesis of the products described herein represents a marked advantage for the large scale preparation of these compounds. In general, the derivatives of the present invention may be readily obtained through sequences recognized by those knowledgeable in the art as straightforward. These sequences are presented in schemes 1 to 7 discussed below.
Scheme 1 illustrates a generic example for the preparation of a key intermediate needed for the synthesis of HIV protease inhibitors.
Note:
a) For scheme 1, R1 represents an alkyl or cycloalkylalkyl side chain as defined above
b) R2 represents a benzenesulfonyl group of formula III, a thiophenesulfonyl group of formula IV, a 1-naphthylsulfonyl, a 2-naphthylsulfonyl or a 8-quinolinesulfonyl group as defined above
As shown in scheme 1, the Nxcex1,Nxcex1-disubstituted L-lysine derivative 5 was obtained from commercially available L-lysine 1 in a four-step reaction sequence. This preparation uses the cyclic form of L-lysine in order to manipulate the Nxcex1-amino group without the need for protective groups. First, L-lysine was transformed into L-xcex1-amino-xcex5-caprolactam 2 upon treatment with hydrochloric acid in methanol followed by neutralization with sodium hydroxide. The caprolactam 2 is also commercially available. Reductive alkylation of derivative 2 with an appropriate aldehyde and NaBH(OAc)3 in dichloroethane lead to the Nxcex1-alkylamino-xcex5-caprolactam 3. Then, sulfonation with an arylsulfonyl chloride or a substituted-arylsulfonyl chloride in the presence of triethylamine in dichloromethane gave compound 4 in excellent yields. The Nxcex1,Nxcex1-disubstituted L-lysine derivative 5 was obtained quantitatively by acid hydrolysis of the cyclic amide 4. 
Scheme 2 illustrates the preparation of HIV protease inhibitors bearing either a carboxylic function, compound 6, or an alcohol function, compound 8, on the final product. In other words, this scheme shows the synthesis of a L-lysine derivative or a (2S) 2,6-diaminohexanol derivative
Note:
a) For scheme 2, R1 and R2 are as defined above
b) R3 represents H, a straight or branched alkyl group of 1 to 6 carbon atoms, a phenyl or a benzyl group
c) R4 is as defined above
d) R5 represents an amino acid side chain as defined above
Following the indications summarized in Scheme 2, derivative 5 is linked with a substituted amino acid using N,N-carbonyldiimidazole as the activating reagent to yield derivative 6 in good to excellent yields. The various N-acylated (or N-sulfonated) amino acids needed for the coupling reaction were prepared from the appropriate amino acid and acyl chloride (or sulfonyl chloride) using the Schotten-Baumen procedure. Alternatively, derivative 5 is treated with trimethylsilyl chloride in methanol (HCl generated in situ) and the resulting methyl ester intermediate is reduced with lithium aluminum hydride (LAH) in THF to afford 7 in good yields. The (2S) 2,6-diaminohexanol derivative 7 is linked to a substituted amino acid derivative as it is described above for the synthesis of derivative 6. 
The L-lysine divative 6 can be further transformed into a variety of esters 9 as well as amide derivative 10 as shown in scheme 3. These transformations are done under standard reaction conditions. For example, the synthesis of ester 9 can be achieved upon activation of the acid 6 with DCC in the presence of a catalytic amount of N,N-dimethylaminopyridine and an alcohol. The amide 10 can be obtained as described earlier for the preparation of compound 6, see scheme 2.
Note:
a) For scheme 3, R1, R2, R3, R4 and R5 are as described above
b) R10 and R11, same or different, represent an H or a straight or branched alkyl group of 1 to 4 carbon atoms. 
Scheme 4 presents a second approach for the preparation of HIV protease inhibitors of formula 6 and 8. It proceeds by using commercially available Ne-benzyloxycarbonyl-L-lysine methyl ester hydrochloride (11) as the starting material. Reductive alkylation of derivative 11 with an appropriate aldehyde and sodium cyanoborohydride provided the derivative 12. Then, sulfonation with benzenesulfonyl chloride (or substituted-benzenesulfonyl chloride) in the presence of triethylamine (or diisopropylethylamine) in dichloromethane gave compound 13 in excellent yields for the two first steps. Removal of the benzyloxycarbonyl group (Z group) by hydrogen gas in presence of 10% Pd/C yielded the free Nxcex5-amino derivative 14 quantitatively. Acylation of 14 with a substituted amino acid N-hydroxysuccinimide ester provided derivative 15 in excellent yields. The desired HIV protease inhibitors 6 and 8 are easily obtained from the methyl ester 15 by hydrolysis with sodium hydroxide in a mixture of THF and methanol giving the acid 6 or by reduction with LAH giving the alcohol 8, both in excellent yields. It is noteworthy that, under basic hydrolysis of 15 to produce compound 6, some racemization may occur. However, it is not the case when compound 15 is reduced with LAH to give derivative 8.
Note:
a) For scheme 4, R1, R2, R3, R4 and R5 are as described above 
Scheme 5 illustrates the preparation of an anti-protease derivative using a solid phase methodology in accordance with the present invention (see example 21). Any suitable solid phase substrate could be used in such preparation (K. Burgess, Solid phase organic synthesis, Wiley-Interscience, 2000).
Note:
a) For scheme 4, R1 is iso-butyl, R2 is 4-methylbenzenesulfonyl, R3, R4 and R5 are as described above
This process allows the introduction of pharmacophores to a Nxcex1,Nxcex1-disubstituted-L-lysine derivative (such as 16) via the N-terminal function. Thus, in scheme 5, Nxcex1-isobutyl-Nxcex1-(4-methylbenzenesulfonyl)-Nxcex5-(9-fluorenylmethoxycarbonyl)-L-lysine 16 is immobilized on a p-benzyloxybenzylalcohol resin (Wang resin) in DMF for a period of 16 h. The resulting component 17 contained 0.28 meq. of L-lysine derivative/g of resin. At this stage, after removal of the Fmoc protective group under standard reaction conditions (30% piperidine in DMF see T. W. Greene and P. G. M. Wuts, Protective groups in Organic Synthesis, 3rd Edition, John Wiley and Sons, Inc. 2000), the resin can be coupled with a variety of N-acylated (or N-sulfonated) amino acids to give component 18. The N-acylated (or N-sulfonated) amino acids are activated with N-hydroxysuccinimide and DCC in DMF. Cleavage of the resin with TFA in CH2Cl2 leads to the desired L-lysine derivative 19. 
Scheme 6 illustrates the preparation of substituted glycine derivatives used for the synthesis of several HIV protease inhibitors in accordance with the present invention (see examples 114 and 158 below for specific descriptions of the synthesis of such glycine derivatives):
In scheme 6a), N-phenylglycine 20 is treated with an excess butyllithium to give the dianion intermediate to which an appropriate alkyl halide (or arylalkyl halide or tosylate) is added and reacted for a period of 16 h. The final products 21 are obtained in good to excellent yields. An appropriate alkyl halide is defined as bearing a R3 component which can sustain strong basic reaction conditions.
In scheme 6b), methyl bromoacetate 22 is treated with benzylamine in CH2Cl2 at room temperature for 16 h. The N-benzylglycine methyl ester derivative 23 was obtained in 86% yield. This intermediate can be either acylated with a carboxylic acid derivative and DCC in THF or sulfonated with an appropriate sulfonyl chloride and triethylamine in CH2Cl2 to give derivative 24 or 25 as desired in good to excellent yields. 
Scheme 7 shows another methodology for the formation of Nxcex5-substituted-glycyl-L-lysine HIV protease inhibitors via the Nxcex5-iodoacetyl-L-lysine derivative 26 (see example 105 for the detailed description of the synthesis of derivative 26 and its use). Thus, Nxcex1,Nxcex1-disubstituted-L-lysine derivative 5 potassium salt is initially treated with chloroacetyl chloride in the presence of DIEA in THF to give the Nxcex1,Nxcex1-disubstituted-Nxcex5-chloroacetyl-L-lysine intermediate. This intermediate is transformed into the iodoacetyl derivative 26 upon treatment with sodium iodide in dry acetone. Compound 26 is then heated at reflux with a primary (or secondary) amine in the presence of of DIEA in THF to yield the desired Nxcex5-substituted-glycyl-L-lysine derivative 6. In scheme 7, a primary amine is used so R3=H and R5=H. 
As it can be appreciated by the skilled artisan, the above synthetic schemes are not intended to be a comprehensive list of all means by which the compounds described and claimed in this application may be synthesized. Further methods will be evident to those of ordinary skill in the art.
The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
As discussed above, the novel compounds of the present invention are excellent ligands for aspartyl proteases, particularly HIV-1 protease. Accordingly, these compounds are capable of targeting and inhibiting late stage events in the replication, i.e. the processing of the viral polyproteins by HIV encoded protease. Compounds according to this invention advantageously inhibit the ability of the HIV-1 virus to infect immortalized human T cells over a period of days, as determined by an assay measuring the amount of extracellular p24 antigenxe2x80x94a specific marker of viral replication (see, Meek et al., Nature, 343, pp. 90-92 (1990)).
In addition to their use in the prophylaxis or treatment of HIV or HTLV infection, the compounds according to this invention may also be used as inhibitory or interruptive agents for other viruses which depend on aspartyl proteases, similar to HIV or HTLV aspartyl proteases, for obligatory events in their life cycle. Such compounds inhibit the proteolytic processing of viral polyprotein precursors by inhibiting aspartyl protease. Because aspartyl protease is essential for the production of mature virions, inhibition of that processing effectively blocks the spread of virus by inhibiting the production and reproduction of infectious virions, particularly from acutely and chronically infected cells. The compounds of this invention advantageously inhibit aspartyl proteases, thus blocking the ability of aspartyl proteases to catalyse the hydrolysis of peptide bonds.
The compounds of this invention may be employed in a conventional manner for the treatment or prevention of HIV, HTLV, and other viruses, which depend on aspartyl proteases for obligatory events in their life cycle. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques. For example, a compound of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a virally infected patient in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of the viral infection.
Alternatively, the compounds of this invention may be used in vaccines and methods for protecting individuals against viral infection over an extended period of time. The compounds may be employed in such vaccines either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of protease inhibitors in vaccines. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants conventionally employed in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against viral infections, such as HIV infection. As such, the novel protease inhibitors of this invention can be administered as agents for treating or preventing viral infections, including HIV infection, in a mammal.
The compounds of this invention may be administered to a healthy or HIV-infected patient either as a single agent or in combination with other antiviral agents which interfere with the replication cycle of HIV. By administering the compounds of this invention with other antiviral agents which target different events in the viral life cycle, the therapeutic effect of these compounds is potentiated. For instance, the co-administered antiviral agent can be one which targets early events in the viral life cycle, such as attachment to the cell receptor and cell entry, reverse transcription and viral DNA integration into cellular DNA. Antiviral agents targeting such early life cycle events include among others polysulfated polysaccharides, sT4 (soluble CD4) and other compounds which block binding of virus to CD4 receptors on CD4 bearing T-lymphocytes and other CD4(+) cells, or inhibit fusion of the viral envelope with the cytoplasmic membrane, and didanosine (ddI), zalcitabine (ddC), stavudine (d4T), zidovudine (AZT) and lamivudine (3TC) which inhibit reverse transcription. Other anti-retroviral and antiviral drugs may also be co-administered with the compounds of this invention to provide therapeutic treatment for substantially reducing or eliminating viral infectivity and the symptoms associated therewith. Examples of other antiviral agents include ganciclovir, dideoxycytidine, trisodium phosphonoformate, eflornithine, ribavirin, acyclovir, alpha interferon and trimenotrexate. Additionally, other types of drugs may be used to potentiate the effect of the compounds of this invention, such as viral uncoating inhibitors, inhibitors of Tat or Rev trans-activating proteins, antisense molecules or inhibitors of the viral integrase. These compounds may also be co-administered with other inhibitors of HIV aspartyl protease.
Combination therapies according to this invention exert a synergistic effect in inhibiting HIV replication because each component agent of the combination acts on a different site of HIV replication. The use of such combinations also advantageously reduces the dosage of a given conventional anti-retroviral agent that would be required for a desired therapeutic or prophylactic effect as compared to when that agent is administered as a monotherapy. These combinations may reduce or eliminate the side effects of conventional single anti-retroviral agent therapies while not interfering with the anti-retroviral activity of those agents. These combinations reduce the potential of resistance to single agent therapies, while minimizing any associated toxicity. These combinations may also increase the efficacy of the conventional agent without increasing the associated toxicity. Preferred combination therapies include the administration of a compound of this invention with AZT, 3TC, ddI, ddC, d4T or other reverse transcriptase inhibitors.
Alternatively, the compounds of this invention may also be co-administered with other HIV protease inhibitors such as Ro 31-8959 (Saquinavir; Roche), L-735,524(Indinavir; Merck), AG-1343 (Nelfinavir; Agouron), A-84538 (Ritonavir; Abbott), ABT-378/r (Lopinavir; Abbott), and VX-478 (Amprenavir; Glaxo) to increase the effect of therapy or prophylaxis against various viral mutants or members of other HIV quasi species.
We prefer administering the compounds of this invention as single agents or in combination with retroviral reverse transcriptase inhibitors, or other HIV aspartyl protease inhibitors. We believe that the co-administration of the compounds of this invention with retroviral reverse transcriptase inhibitors or HIV aspartyl protease inhibitors may exert a substantial synergistic effect, thereby preventing, substantially reducing, or completely eliminating viral infectivity and its associated symptoms.
The compounds of this invention can also be administered in combination with immunomodulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyldithiocarbamate sodium, tumor necrosis factor, naltrexone and rEPO) antibiotics (e.g., pentamidine isethionate) or vaccines to prevent or combat infection and disease associated with HIV infection, such as AIDS and ARC.
When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this invention may be comprised of a combination of an aspartyl protease inhibitor of this invention and another therapeutic or prophylactic agent.
Although this invention focuses on the use of the compounds disclosed herein for preventing and treating HIV infection, the compounds of this invention can also be used as inhibitory agents for other viruses that depend on similar aspartyl proteases for obligatory events in their life cycle. These viruses include, but are not limited to, retroviruses causing AIDS-like diseases such as simian immunodeficiency viruses, HIV-2, HTLV-I and HTLV-II. In addition, the compounds of this invention may also be used to inhibit other aspartyl proteases and, in particular, other human aspartyl proteases including renin and aspartyl proteases that process endothelin precursors.
Pharmaceutical compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethyleneglycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of this invention may be administered orally, parenterally by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are amino acid, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv. or a similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspension and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene or polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable neat formulation. Topically-transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Dosage levels of between about 0.01 and about 25 mg/kg body weight per day, preferably between about 0.5 and about 25 mg/kg body weight per day of the active ingredient compound are useful in the prevention and treatment of viral infection, including HIV infection. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.
Upon improvement of a patient""s condition, a maintenance dose of a compound, composition or combination of this invention may be administered if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis, upon any recurrence of disease symptoms.
As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the infection, the patient""s disposition to the infection and the judgment of the treating physician.
The compounds of this invention are also useful as commercial reagents which effectively bind to aspartyl proteases, particularly HIV aspartyl protease. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide by an aspartyl protease, or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial aspartyl protease inhibitors will be evident to those of ordinary skill in the art.
Enzymatic Assay for Determining the Inhibition Constant (Ki) of Synthetic Compounds Targeting the HIV Protease
This is a fluorometric assay based on the cleavage by protease of a substrate carrying a donor group (EDANS) and an acceptor group (DABCYL) on each side of the cleavage site, interacting together through fluorescence resonance energy transfer (FRET) as described by Matayoshi et al. (Science 247:954-954, 1990).
After calculation of Vo and Vi, the inhibition constant (Ki) of the compound is determined using the equation of Henderson:       Vo    Vi    =            1      +                                    [            I            ]                                Ki            app                          ⁢                  xe2x80x83                ⁢        Where        ⁢                  xe2x80x83                ⁢        Ki              =                  Ki        app                              1          +                      [            S            ]                          Km            
where
Vo=the enzyme""s initial velocity
Vi=the enzyme velocity in the presence of the inhibitory compound,
[I]=inhibitor concentration,
[S]=substrate concentration,
Km=Michaelis-Menten constant and
Kiapp=apparent Ki
Graphs are traced and the Ki determined using GraphPad Prism software v. 3.0.
The compounds listed in Tables 1 and 2 were prepared by following Scheme 1, 2, 3, 4, 5, 6 or 7; the numbers of the compounds listed in the table correspond to the example numbers presented in the experimental section (see examples below). The activities of the compounds are also listed in the same tables demonstrating their potential usefulness. In Table 1 are shown compounds of formula I wherein Y, n, Cx, R1, R2, R3, R4 and R5 are as presented in Table 1. In Table 2 are shown compounds of formula II wherein Y, n, Cx, R1, R2, R4 and Ra are as presented in Table 2.